New approach for preparation of 2,3,7-trisubstituted 3,4-dihydroisoquinolinone libraries on solid phase.

In an attempt to prepare 7-substituted 3,4-dihydroisoquinolinone family of compounds, we observed an unexpected decarboxylation. The reaction of 4-nitrohomophthalic anhydride with a Schiff base formed on solid support leads to the formation of core structure. LC-MS and 1H NMR analysis confirmed the structure of unexpected intermediate. A library of 38,400 compounds was produced using this new synthetic approach.

Combinatorial chemistry reveals a new motif that binds the platelet fibrinogen receptor, gpIIbIIIa.

Among cell adhesion molecules, the classic Arg-Gly-Asp (RGD) motif is the best studied. We used combinatorial chemical and affinity immunochemical methods to find a novel motif of unnatural peptide ligands for the fibrinogen receptor of platelets, gpIIbIIIa (alphaIIbbeta3). The new d-amino acid motif, p(f/y)l, is unique among the ligands that bind the RGD pocket: It lacks the carboxylic acid group that is believed to coordinate with calcium in the MIDAS motif of the receptor. With an IC50 of 14 microM for the most potent compound, these linear p(f/y)l peptides had affinities similar to those of linear peptides containing RGD, and reversed sequences failed to compete with binding up to 1 mM. As the new motif was so different, molecular modeling was employed to suggest a model for molecular recognition. A reversed binding mechanism common for d-amino acid mimics of natural l-amino acid peptides offers an attractive hypothesis that suggests three points of contact similar to those made by the RGD-mimicking monoclonal antibody, OPG2. Interestingly, the model proposes that pi-electrons in the new motif may substitute for the carboxylate group present in all other RGD-types of ligands. Although modeling linear peptides is subjective, the pi-bonding model provides intriguing possibilities for medicinal chemistry after appropriate confirmatory studies.

The domino blocks: a simple solution for parallel solid-phase organic synthesis.

The domino block is a reaction block for manual and semi-automatic parallel solid-phase organic synthesis that simplifies liquid exchange and integrates common synthetic steps. The domino block consists of enclosed reaction vessels, polypropylene syringes, attached to a manifold that clamps the syringes and connects them to a common port. Liquid is removed from the closed reaction vessels by vacuum connected to the common port. The vacuum formed inside each reaction vessel is subsequently used to draw fresh solvent or reagent into each reaction vessel.

Semi-automated high throughput combinatorial solid-phase organic synthesis.

A semi-automated technique for massive parallel solid-phase organic synthesis based on a "split only" strategy is described. Two different types of purpose-oriented reaction vessels are used. The initial steps are performed in domino blocks, and the resin-bound intermediates then split into wells of a micro plate for the last combinatorial step. The domino block is a reaction block for manual and semi-automatic parallel solid-phase organic synthesis that simplifies liquid exchange and integrates common synthetic steps. The synthesis in micro plates does not use any filter for separation of resin beads from the supernatant liquid, and allows high throughput parallel synthesis on solid phase to be performed. This technique, documented on examples of diverse disubstituted benzenes, includes the use of gaseous cleavage in the last synthetic step and allows the synthesis of thousands of compounds per day in mg quantities.

High-volume cellular screening for anticancer agents with combinatorial chemical libraries: a new methodology.

A single-step cancer cell cytotoxic assay system for anticancer drug discovery has been developed which facilitates rapid screening of large combinatorial chemical libraries synthesized using the 'one-bead-one-compound' (OBOC) methodology. Each OBOC library bead incorporates two orthogonally cleavable linkers that release the bead-bound compound at a different pH. The assay utilizes high concentrations of tumor cells mixed directly with OBOC beads and plated in soft agarose containing tissue culture medium. One of the orthogonal linkers is cleaved at neutral pH in tissue culture releasing an aliquot of compound to diffuse at a relatively high local concentration into the soft agarose immediately surrounding the bead. Active compounds are identified visually from a clear ring of tumor cell lysis which forms within 48 h around just the rare bead releasing a cytotoxic compound. The bead releasing a cytotoxin is then plucked from the agar and the remaining compound still linked to the bead can be released for structural analysis, followed by compound resynthesis and confirmatory testing. This assay system has been successfully applied to identification of lead cytotoxic compounds from model peptidic and non-peptidic combinatorial chemical libraries. Use of this methodology may facilitate anticancer drug discovery.

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

A One-Bead One-Peptide Combinatorial Library Method for B-Cell Epitope Mapping

The one-bead one-peptide combinatorial library method represents a powerful approach to the discovery of binding peptides for various macromolecular targets. It involves the synthesis of millions of peptides on beads such that each bead displays only one peptide entity. The peptide-beads that interact with a specific macromolecular target are then isolated for structure determination. We have applied this method to discovering peptide ligands for several murine monoclonal antibodies: (i) anti-beta-endorphin (continuous epitope), (ii) anti-vmos peptide, (iii) anti-human insulin (discontinuous epitope), and (iv) surface immunoglobulins (μkappa) of two murine B-cell lymphoma cell lines (antigen unknown).

Structurally homogeneous and heterogeneous synthetic combinatorial libraries.

We have designed and synthesized structurally homogeneous and heterogeneous nonpeptide libraries. Structurally homogeneous libraries are characterized by the presence of one common structural unit, a scaffold, in all library compounds (e.g. cyclopentane, cyclohexane, diketopiperazine, thiazolidine). In structurally heterogeneous libraries different organic reactions (acylation, etherification, reductive amination, nucleophilic displacement) were applied to connect bifunctional building blocks unrelated in structure (aromatic hydroxy acids, aromatic hydroxy aldehydes, amino alcohols, diamines, and amino acids). The focus of this communication is to document the use of bifunctional building blocks for the design and synthesis of structurally heterogeneous libraries of N-(alkoxy acyl)amino acids, N,N'-bis-(alkoxy acyl)diamino acids, N-acylamino ethers, N-(alkoxy acyl)amino alcohols, N-alkylamino ethers, and N-(alkoxy aryl)diamines.

Bifunctional scaffolds as templates for synthetic combinatorial libraries.

A small-molecule synthetic combinatorial library was designed and synthesized that features potential pharmacophores attached to a variety of small cyclic scaffolds. The synthesis of the library involved randomization of three types of building blocks: 20 amino acids, 10 aromatic hydroxy acids and 21 alcohols, totaling a library complexity of 4200 compounds. Mitsunobu polymer-supported etherification was used in the last randomization. The library compounds were attached to beads via an ester-bond linkage enabling both on-bead as well as in-solution screening. When the library was tested against a model target, streptavidin, specific binders were found. The structures of the most active compounds were determined from the fragmentation pattern in MS/MS experiments.

Synthetic library techniques: subjective (biased and generic) thoughts and views.

Various aspects of synthetic diversity generation and screening are discussed. Controversial issues are raised and different points of view are presented. We hope the article will stimulate thinking about the utilization of library techniques and start a discussion about questions concerning their application.

MARS--multiple automated robotic synthesizer for continuous flow of peptides.

We have designed and constructed a multiple automated robotic synthesizer, the MARS. Its novel timing procedure for handling multiple synthetic tasks eliminates unnecessary respite time by keeping the robotic arm in continuous operation. Polypropylene syringes equipped at the bottom with polypropylene frits serve as physically independent reaction vessels. All operations are performed by the robotic arm, which is equipped with a specially designed gripper to hold a syringe and to aspirate and dispense liquid. Typically, the MARS synthesizes concurrently 5 to 15 peptides of different length, and once one peptide is finished it automatically starts the synthesis of the next peptide in the queue, assuring a continuous flow of peptides.

Linear presentation of variable side-chain spacing in a highly diverse combinatorial library.

A synthetic library that presents potential pharmacophores in a linear fashion with variable spacing was designed (alpha, beta, gamma-library). To prove the concept, we synthesized a number of individual compounds as well as a model library. Diamino acids connected by amide bonds via their alpha- or side-chain amino groups were used to form the backbone (scaffold) of this library. The remaining amino group of the diamino acids were acylated by a variety of carboxylic acids, generating an appreciable diversity of compounds in this library. The compositions of compounds in the library were identified by reading a peptide tag synthesized concurrently with the library structures. This code contained the information regarding the carboxylic acid coupled, and the diamino acid and amino group to which the acid was coupled.

Library of libraries: approach to synthetic combinatorial library design and screening of "pharmacophore" motifs.

Construction of synthetic combinatorial libraries is described that allows for the generation of a library of motifs rather than a library of compounds. Peptide libraries based on this strategy were synthesized and screened with model targets streptavidin and anti-beta-endorphin antibody. The screens resulted in observation of expected motifs providing evidence of the effectiveness of the suggested approach.

Conformation, pore-forming activity, and antigenicity of synthetic peptide analogues of a spiralin putative amphipathic alpha helix.

Current models depict spiralin as a bitopic transmembrane protein with the transbilayer domain being an amphipathic alpha helix. However, though secondary structure prediction methods suggest a helical conformation for the hypothetical transmembrane segment of spiralin, no potential transmembrane helices could be detected in this protein using the method of Von Heijne (Von Heijne, G. (1992) J. Mol. Biol. 225, 487-494). Therefore, we have reconsidered the spiralin topological model by investigating the properties of the chemically synthesized peptides SM-BC3 (LNAVNTYATLAKAVLDAIQN-NH2) and SC-R8A2 (LNAVNTYATLASAVLEAIKN-NH2), corresponding to the hypothetical transmembrane segments of spiralins of two distinct spiroplasma species. The hydrophobic moment plot method suggests that these spiralin amino acid stretches are class G amphipathic alpha helices (i.e., helices localized on the surface of a globular protein domain). Circular dichroism spectra showed that both peptides have little ordered structure in aqueous solutions but adopt a mainly helical conformation in the presence of 25% trifluoroethanol or in detergent micelles (up to 74% alpha helix). Both peptides formed concentration- and voltage-dependent pores in planar lipid bilayers with a unitary conductance of 130 pS in 1 M KCl and with mean numbers of monomers per conducting aggregates of 6 for SC-R8A2 and 9 for SM-BC3. However, the two peptides displayed a haemolytic activity only at high concentrations (> 250 microM) and reacted with antibodies raised against membrane-bound spiralin. Together with previously published results, these data suggest that spiralin is a monotopic membrane protein anchored at the surface of the spiroplasma cell and that the 20-residue amphipathic segment is most probably a class G helix containing a B-cell epitope.

Neutralizing antibodies against highly cytopathic Zairian human immunodeficiency type-1 virus (HIV-1) NDK are present in sera outside Africa.

The prototype virus HIV-1 LAV and highly cytopathic Zairian virus HIV-1 NDK belong to the genetic subtypes B and D and represent low and highly cytopathic phenotypes, respectively. Their neutralization pattern and serotype were studied with respect to differences in their genotypes and phenotypes. Sera from HIV-1-infected persons living in four geographically distant areas, Philadelphia (USA), Ribeirao Preto (Brazil), Marseille (France) and Kinshasa (Zaire), were tested for the presence of type-specific and group-specific cross-reacting neutralizing antibodies against HIV-1 LAV and HIV-1 NDK in a continuous cell line MT4. The majority of type-specific antibodies were directed against HIV-1 LAV in Philadelphia, Ribeirao Preto and Marseille, and against HIV-1 NDK in Kinshasa. However, some sera with an HIV-1 NDK type-specific neutralization pattern were also found in Philadelphia, Ribeirao Preto and Marseille. These results indicate that strains with an HIV-1 NDK-like serotype could be found outside Africa. The presence of type-specific neutralizing antibodies against HIV-1 NDK in sera from North and South America and Europe should be taken into account during attempts to serotype HIV as well as in the course of selection of HIV-1 candidate strains for an AIDS vaccine.

One-bead-one-structure combinatorial libraries.

Combinatorial libraries employing the one-bead-one-compound technique are reviewed. Two distinguishing features characterize this technique. First, each compound is identified with a unique solid support, enabling facile segregation of active compounds. Second, the identity of a compound on a positively reacting bead is elucidated only after its biological relevance is established. Direct methods of structure identification (Edman degradation and mass spectroscopy) as well as indirect "coding" methods facilitating the synthesis and screening of nonpeptide libraries are discussed. Nonpeptide and "scaffold" libraries, together with a new approach for the discovery of a peptide binding motif using a "library of libraries," are also discussed. In addition, the ability to use combinatorial libraries to optimize initially discovered leads is illustrated with examples using peptide libraries.

Adjuvant effect of liposomes and adamantylamide dipeptide on antigenicity of entrapped synthetic peptide derived from HIV-1 transmembrane region glycoprotein gp41.

The synthetic peptide antigen (Ag) (the primary structure Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile- Cys-Thr derived from the envelope glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) and exerting specificity with all HIV-1-positive sera available in the Czech Republic (and also in a panel of 10,000 sera from WHO)) was conjugated with bovine serum albumin (BSA) and encapsulated into liposomes. Adjuvant activities of liposomes with various lipid compositions were compared with Freund's complete adjuvant (FCA) and with aluminium hydroxide (AL). The immune response to BSA-Ag liposomes with coentrapped adamantylamide dipeptide (AdDP) was comparable with that of FCA in terms of longevity and levels of specific antibodies in mouse sera.